Phorboxazoles are natural products isolated from the marine sponge Phorbas sp. The isolation, partial structure determination, and cytostatic activity towards the US National Cancer Institute's panel of 60 human cancer lines (mean GI50 values of less than 1.6 x 10-9 M; most cell lines were 100% inhibited at this lowest test concentration) were reported in August, 1995. The phorboxazoles represent an entirely new class of cytostatic agents, and are "among the most potent cytostatic agents yet discovered." The phorboxazoles' mechanism of action is not known, and additional material is unavailable from natural sources. The phorboxazoles have primary cellular targets and a mechanism of action that are distinct from those of known chemotherapeutic agents. Total synthesis represents an alternative source of the natural product and structural analogs. Synthetic phorboxazole A and several close synthetic analogs are cytotoxic in the pM concentration range and induce apoptotic cell death. The original synthetic entry has allowed some preliminary structure-activity relationship data to be obtained with respect to anticancer activity, and the existing synthesis may be modified to generate probes for the identification of the cellular targets. The overall goal of this proposal is to continue to develop the phorboxazole chemotype as a promising new class of anticancer therapeutic agents by innovating further synthetic organic chemistry to uniquely and rapidly access structural analogs of the phorboxazoles, identify their cellular targets, and determine the essential phorboxazole pharmacophore and likely mode of action. Identification of the phorboxazole receptor will be undertaken using affinity derivatives of synthetic phorboxazole A that will initially be screened against phage displayed libraries of human cDNA. This will be followed by classic affinity methods, as necessary. A novel fragment coupling approach to access the phorboxazole molecular architecture and in vitro screens will be used to test specific structure-activity hypotheses, map the natural product's essential pharmacophore and identify novel molecules that are significantly simpler in structure but maintain the anticancer activity of the parent compounds. The successful outcome of this project will provide unique, synthetically accessible phorboxazole-related anticancer agents. Identification of cellular targets of the phorboxazoles will provide direct insight into the mode of action and may provide a previously unexplored target for therapy.